RESUMO
Osteoarthritis belongs to the most common joint diseases in humans and animals and shows increased incidence in older patients. The bioactivities of collagen hydrolysates, sulfated glucosamine and a special fatty acid enriched dog-food were tested in a dog patient study of 52 dogs as potential therapeutic treatment options in early osteoarthritis. Biophysical, biochemical, cell biological and molecular modeling methods support that these well-defined substances may act as effective nutraceuticals. Importantly, the applied collagen hydrolysates as well as sulfated glucosamine residues from marine organisms were strongly supported by both an animal model and molecular modeling of intermolecular interactions. Molecular modeling of predicted interaction dynamics was evaluated for the receptor proteins MMP-3 and ADAMTS-5. These proteins play a prominent role in the maintenance of cartilage health as well as innate and adapted immunity. Nutraceutical data were generated in a veterinary clinical study focusing on mobility and agility. Specifically, key clinical parameter (MMP-3 and TIMP-1) were obtained from blood probes of German shepherd dogs with early osteoarthritis symptoms fed with collagen hydrolysates. Collagen hydrolysate, a chondroprotective food supplement was examined by high resolution NMR experiments. Molecular modeling simulations were used to further characterize the interaction potency of collagen fragments and glucosamines with protein receptor structures. Potential beneficial effects of collagen hydrolysates, sulfated glycans (i.e., sulfated glucosamine from crabs and mussels) and lipids, especially, eicosapentaenoic acid (extracted from fish oil) on biochemical and physiological processes are discussed here in the context of human and veterinary medicine.
Assuntos
Cartilagem Articular/efeitos dos fármacos , Colágeno/farmacologia , Dieta/veterinária , Suplementos Nutricionais , Doenças do Cão/dietoterapia , Osteoartrite/veterinária , Substâncias Protetoras/farmacologia , Animais , Organismos Aquáticos , Colágeno/química , Colágeno/uso terapêutico , Cães , Osteoartrite/dietoterapia , Substâncias Protetoras/química , Substâncias Protetoras/uso terapêuticoRESUMO
Second-derivative absorption spectroscopy was employed to monitor the response of effective symmetry of cytochromes a and a3 to the redox and ligation states of bovine cytochrome c oxidase (CcO). The Soret band π â π* electronic transitions were used to display the changes in symmetry of these chromophores induced by the reduction of CcO inhibited by the exogenous ligands and during catalytic turnover. The second derivative of the difference absorption spectra revealed only a single Soret band for the oxidized cytochromes a and a3 and cyanide-ligated oxidized cytochrome a3. In contrast, two absorption bands were resolved in ferrous cytochrome a and ferrous cytochrome a3 ligated with cyanide. A transition from one-band spectrum to two-band spectrum indicates the lowering of symmetry of these hemes due to the alteration of their immediate surroundings. It is suggested that the changes in polarity occurring in the vicinity of these cofactors are main reason for the split of the Soret band of both ferrous cytochrome a and cyanide-bound ferrous cytochrome a3.
Assuntos
Citocromos a3/metabolismo , Citocromos a/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Heme/metabolismo , Animais , Bovinos , Cianetos/química , Cianetos/metabolismo , Citocromos a/química , Citocromos a3/química , Complexo IV da Cadeia de Transporte de Elétrons/química , Elétrons , Heme/química , OxirreduçãoRESUMO
Flavin mononucleotide (FMN) is a highly efficient photosensitizer (PS) yielding singlet oxygen (1 O2 ). However, its 1 O2 production efficiency significantly decreases upon isoalloxazine ring encapsulation into the protein matrix in genetically encoded photosensitizers (GEPS). Reducing isoalloxazine ring interactions with surrounding amino acids by protein engineering may increase 1 O2 production efficiency GEPS, but at the same time weakened native FMN-protein interactions may cause undesirable FMN dissociation. Here, in contrast, we intentionally induce the FMN release by light-triggered sulfur oxidation of strategically placed cysteines (oxidation-prone amino acids) in the isoalloxazine-binding site due to significantly increased volume of the cysteinyl side residue(s). As a proof of concept, in three variants of the LOV2 domain of Avena sativa (AsLOV2), namely V416C, T418C, and V416C/T418C, the effective 1 O2 production strongly correlated with the efficiency of irradiation-induced FMN dissociation (wild type (WT) < V416C < T418C < V416C/T418C). This alternative approach enables us: (i) to overcome the low 1 O2 production efficiency of flavin-based GEPSs without affecting native isoalloxazine ring-protein interactions and (ii) to utilize AsLOV2, due to its inherent binding propensity to FMN, as a PS vehicle, which is released at a target by light irradiation.
Assuntos
Flavoproteínas , Fármacos Fotossensibilizantes , Flavoproteínas/química , Flavoproteínas/metabolismo , Domínios Proteicos , Sítios de Ligação , Aminoácidos , Mononucleotídeo de Flavina/químicaRESUMO
The global outbreak of SARS-CoV-2/COVID-19 provided the stage to accumulate an enormous biomedical data set and an opportunity as well as a challenge to test new concepts and strategies to combat the pandemic. New research and molecular medical protocols may be deployed in different scientific fields, e.g., glycobiology, nanopharmacology, or nanomedicine. We correlated clinical biomedical data derived from patients in intensive care units with structural biology and biophysical data from NMR and/or CAMM (computer-aided molecular modeling). Consequently, new diagnostic and therapeutic approaches against SARS-CoV-2 were evaluated. Specifically, we tested the suitability of incretin mimetics with one or two pH-sensitive amino acid residues as potential drugs to prevent or cure long-COVID symptoms. Blood pH values in correlation with temperature alterations in patient bodies were of clinical importance. The effects of biophysical parameters such as temperature and pH value variation in relation to physical-chemical membrane properties (e.g., glycosylation state, affinity of certain amino acid sequences to sialic acids as well as other carbohydrate residues and lipid structures) provided helpful hints in identifying a potential Achilles heel against long COVID. In silico CAMM methods and in vitro NMR experiments (including 31P NMR measurements) were applied to analyze the structural behavior of incretin mimetics and SARS-CoV fusion peptides interacting with dodecylphosphocholine (DPC) micelles. These supramolecular complexes were analyzed under physiological conditions by 1H and 31P NMR techniques. We were able to observe characteristic interaction states of incretin mimetics, SARS-CoV fusion peptides and DPC membranes. Novel interaction profiles (indicated, e.g., by 31P NMR signal splitting) were detected. Furthermore, we evaluated GM1 gangliosides and sialic acid-coated silica nanoparticles in complex with DPC micelles in order to create a simple virus host cell membrane model. This is a first step in exploring the structure-function relationship between the SARS-CoV-2 spike protein and incretin mimetics with conserved pH-sensitive histidine residues in their carbohydrate recognition domains as found in galectins. The applied methods were effective in identifying peptide sequences as well as certain carbohydrate moieties with the potential to protect the blood-brain barrier (BBB). These clinically relevant observations on low blood pH values in fatal COVID-19 cases open routes for new therapeutic approaches, especially against long-COVID symptoms.
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Pesticide use worldwide exhibits a positive effect on agricultural production while it may negatively affect organisms living in soil, water or the air. Importantly, numerous negative health effects also occur in humans exposed to (accumulated) pesticides or their metabolites over a long period of time. To prevent both environmental catastrophes and adverse human health impacts, initial studies of the selected pesticides need to be performed together with the constant post-approval control; risk assessment analysis and on site monitoring have to be continuously carried out. Given this, Raman spectroscopy, especially surface-enhanced Raman spectroscopy (SERS), during the last decade has become a powerful analytical technique since it can offer quick, selective, and in situ detection of selected pollutants found in analyzed samples at very low concentrations. Moreover, the structural changes caused by the pollutant-biomacromolecule interaction can also be recognized in the molecule-specific Raman spectral signatures of biomolecules. In this study, we report a vibrational characterization of the fungicide molecule Tebuconazole (TB) which is listed to be a possible carcinogen. Even though its international and common use there is no evidence about the use of Raman/SERS spectroscopy to detect it sensitively and selectively as well as to analyse its impacts on biological systems. Therefore, we have recorded and calculated Raman and infrared spectra of TB. Furthermore, SERS spectra of TB were also registered and comprehensively analysed in view of the employed SERS substrates, dependence on the excitation wavelengths and pH of the analysed molecular systems. The molecule of TB interacts preferentially through the triazole moiety with the colloidal metal nanoparticles (NPs) whereas the silver NPs prepared by reduction of silver nitrate with hydroxylamine hydrochloride resulted to be the most effective ones. Consequently, the limit of detection was determined to be 1.4 µM≈430 ppb. The present paper thus could serve significantly for further investigations focused on both conducting vibrational analyses of structurally related molecules as well as providing a more precise explanation of the mechanism of action of TB and its influence on biological macromolecules.
Assuntos
Nanopartículas Metálicas , Praguicidas , Humanos , Análise Espectral Raman , Triazóis , VibraçãoRESUMO
Many types of cancer such as prostate cancer, myeloid leukemia, breast cancer, glioblastoma display strong chemo resistance, which is supported by enhanced expression of multiple anti-apoptotic Bcl-2, Bcl-XL and Mcl-1 proteins. The viable anti-cancer strategies are based on developing anti-apoptotic Bcl-2 proteins inhibitors, BH3 mimetics. Our focus in past years has been on the investigating a new potential BH3 mimetic, Hypericin (Hyp). Hyp is a naturally occurring photosensitive compound used in photodynamic therapy and diagnosis. We have demonstrated that Hyp can cause substantial effects in cellular ultrastructure, mitochondria function and metabolism, and distribution of Bcl2 proteins in malignant and non-malignant cells. One of the possible mechanisms of Hyp action could be the direct interactions between Bcl-2 proteins and Hyp. We investigated this assumption by in silico computer modelling and in vitro fluorescent spectroscopy experiments with the small Bcl2 peptide segments designed to correspond to Bcl2 BH3 and BH1 domains. We show here that Hyp interacts with BH3 and BH1 peptides in concentration dependent manner, and shows the stronger interactions than known BH3 mimetics, Gossypol (Goss) and ABT-263. In addition, interactions of Hyp, Goss and ABT263, with whole purified proteins Bcl-2 and Mcl-1 by fluorescence spectroscopy show that Hyp interacts stronger with the Bcl-2 and less with Mcl-1 protein than Goss or ABT-263. This suggest that Hyp is comparable to other BH3 mimetics and could be explore as such. Hyp cytotoxicity was low in human U87 MG glioma, similar to that of ABT263, where Goss exerted sufficient cytotoxicity, suggesting that Hyp acts primarily on Bcl-2, but not on Mcl-1 protein. In combination therapy, low doses of Hyp with Goss effectively decreased U87 MG viability, suggesting a possible synergy effect. Overall, we can conclude that Hyp as BH3 mimetic acts primarily on Bcl-2 protein and can be explored to target cells with Bcl-2 over-expression, or in combination with other BH3 mimetics, that target Mcl-1 or Bcl-XL proteins, in dual therapy.
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Amphiphilic compounds, both detergents and lipids, are important tools for in vitro analysis of water-soluble and integral membrane proteins. A key question is whether these two groups of amphiphilic molecules use the same pathway to affect structural and functional integrity of proteins. In the present study, we tested the effect of non-ionic detergent dodecyl maltoside (DDM), two phospholipids, 1,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC), 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), and the detergent-phospholipid mixtures on insulin amyloidogenesis in vitro. Amyloidogenesis of insulin is significantly affected by DDM in a time-and dose-dependent manner, but only slightly affected by either of phospholipids. Addition of DHPC or DMPC to detergent does not alter the inhibiting pattern, suggesting that DDM preferably binds to insulin. The molecular modeling revealed that DDM and the phospholipids occupy equivalent binding sites. DDM, due to the presence of maltose with several oxygen atoms (hydroxylic, glycosidic and ring) is involved in more hydrogen bonds than DHPC or DMPC. Hydrophobic interactions are important factors to stabilize both, DDM and phospholipids in their binding sites. Our results indicate that certain detergents (applying DDM as an example) and selected phospholipids are not always interchangeable in their use to investigate the effect of amphiphilic compounds on the behavior of amyloid-prone proteins.
Assuntos
Detergentes , Fosfolipídeos , Amiloide , Proteínas Amiloidogênicas , Dimiristoilfosfatidilcolina , InsulinaRESUMO
Cytochrome c (cyt c), in addition to its function as an electron shuttle in respiratory chain, is able to perform as a pseudo-peroxidase with a critical role during apoptosis. Incubation of cyt c with an excess of hydrogen peroxide leads to a suicide inactivation of the protein, which is accompanied by heme destruction and covalent modification of numerous amino acid residues. Although steady-state reactions of cyt c with an excess of hydrogen peroxide represent non-physiological conditions, they might be used for analysis of the first-modified amino acid in in vivo. Here, we observed oxidation of tyrosine residues 67 and 74 and heme as the first modifications found upon incubation with hydrogen peroxide. The positions of the oxidized tyrosines suggest a possible migration pathway of hydrogen peroxide-induced radicals from the site of heme localization to the protein surface. Analysis of a size of folded fraction of cyt c upon limited incubation with hydrogen peroxide indicates that the early oxidation of amino acids triggers an accelerated destruction of cyt c. Position of channels from molecular dynamics simulation structures of cyt c points to a location of amino acid residues exposed to reactive oxidants that are thus more prone to covalent modification.
Assuntos
Citocromos c/química , Citocromos c/metabolismo , Peróxido de Hidrogênio/farmacologia , Animais , Dicroísmo Circular , Citocromos c/genética , Cavalos , Espectrometria de Massas , Modelos Moleculares , Simulação de Dinâmica Molecular , Oxirredução , Conformação Proteica , Estabilidade Proteica , Proteólise , Tirosina/químicaRESUMO
Three different array formats to study a challenging field of glycomics are presented here, based on the use of a panel of immobilized glycan or lectins, and on in silico computational approach. Glycan and lectin arrays are routinely used in combination with other analytical tools to decipher a complex nature of glycan-mediated recognition responsible for signal transduction of a broad range of biological processes. Fundamental aspects of the glycan and lectin array technology are discussed, with the focus on the choice and availability of the biorecognition elements, fabrication protocols, and detection platforms involved. Moreover, practical applications of both technologies especially in the field of clinical diagnostics are provided. The future potential of a complementary in silico array technology to reveal details of the protein-glycan-binding profiles is discussed here.
Assuntos
Glicômica/métodos , Lectinas/química , Polissacarídeos/química , Animais , Carboidratos/química , Linhagem Celular , Epitopos/química , Escherichia coli/metabolismo , Humanos , Cinética , Ligantes , Modelos Moleculares , Neoplasias/metabolismo , Transdução de SinaisRESUMO
Mitochondrial cytochrome c oxidase (CcO) is a multisubunit integral membrane complex consisting of 13 dissimilar subunits, as well as three to four tightly bound molecules of cardiolipin (CL). The monomeric unit of CcO is able to form a dimer and participate in the formation of supercomplexes in the inner mitochondrial membrane. The structural and functional integrity of the enzyme is crucially dependent on the full subunit complement and the presence of unperturbed bound CL. A direct consequence of subunit loss, CL removal, or its oxidative modification is the destabilization of the quaternary structure, loss of the activity, and the inability to dimerize. Thus, the intimate interplay between individual components of the complex is imperative for regulation of the CcO aggregation state. While it appears that the aggregation state of CcO might affect its conformational stability, the functional role of the aggregation remains unclear as both monomeric and dimeric forms of CcO seem to be fully active. Here, we review the current status of our knowledge with regard to the role of dimerization in the function and stability of CcO and factors, such as subunit composition, amphiphilic environment represented by phospholipids/detergents, and posttranslational modifications that play a role in the regulation of the CcO aggregation state.
Assuntos
Cardiolipinas/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Coração/fisiologia , Subunidades Proteicas/metabolismo , Animais , Humanos , Mitocôndrias/metabolismo , Fosfolipídeos/metabolismoRESUMO
LOV2 (Light-Oxygen-Voltage) domain from Avena sativa phototropin 1 (AsLOV2) belongs to the superfamily of PAS (Per-Arnt-Sim) domains, members of which function as signaling sensors. AsLOV2 undergoes a conformational change upon blue-light absorption by its FMN cofactor. AsLOV2 wild type (wt) is intensively studied as a photo-switchable element in conjugation with various proteins. On the other hand, its variant AsLOV2 with replaced cysteinyl residue C450, which is critical for the forming a covalent adduct with FMN upon irradiation, forms a precursor for some recently developed genetically encoded photosensitizers. In the presented work, we investigated conformational properties of AsLOV2 wt and its variant C450A by circular dichroism, tryptophan and FMN fluorescence, and differential scanning calorimetry in dependence on pH and temperature. We show that both variants are similarly sensitive towards pH of solvent. On the other hand, the mutation C450A leads to a more stable AsLOV2 variant in comparison with the wild type. Thermal transitions of the AsLOV2 proteins monitored by circular dichroism indicate the presence of significant residual structure in thermally-denatured states of both proteins in the pH range from 4 to 9. Both pH- and thermal- transitions of AsLOV2 are accompanied by FMN leaching to solvent. Higher stability, reversibility of thermal transitions, and efficiency of FMN rebinding in the case of C450A variant suggest that the cofactor release may be modulated by suitable mutations in combination with a suitable physicochemical perturbation. These findings can have implications for a design of genetically encoded photosensitizers.
Assuntos
Fototropinas/química , Proteínas de Plantas/química , Substituição de Aminoácidos , Avena/química , Avena/metabolismo , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Mononucleotídeo de Flavina/química , Concentração de Íons de Hidrogênio , Modelos Moleculares , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Espectrometria de Fluorescência , Relação Estrutura-Atividade , Triptofano/químicaRESUMO
Flavin mononucleotide (FMN) belongs to the group of very efficient endogenous photosensitizers producing singlet oxygen, 1O2, but with limited ability to be targeted. On the other hand, in genetically-encoded photosensitizers, which can be targeted by means of various tags, the efficiency of FMN to produce 1O2 is significantly diminished due to its interactions with surrounding amino acid residues. Recently, an increase of 1O2 production yield by FMN buried in a protein matrix was achieved by a decrease of quenching of the cofactor excited states by weakening of the protein-FMN interactions while still forming a complex. Here, we suggest an alternative approach which relies on the blue light irradiation-induced dissociation of FMN to solvent. This dissociation unlocks the full capacity of FMN as 1O2 producer. Our suggestion is based on the study of an irradiation effect on two variants of the LOV2 domain from Avena sativa; wild type, AsLOV2 wt, and the variant with a replaced cysteine residue, AsLOV2 C450A. We detected irradiation-induced conformational changes as well as oxidation of several amino acids in both AsLOV2 variants. Detailed analysis of these observations indicates that irradiation-induced increase in 1O2 production is caused by a release of FMN from the protein. Moreover, an increased FMN dissociation from AsLOV2 wt in comparison with AsLOV2 C450A points to a role of C450 oxidation in repelling the cofactor from the protein.
RESUMO
New analogs of indole phytoalexin 1-methoxyspirobrassinol methyl ether have been designed by replacement of its 2-methoxy group with 2-(substituted phenyl)amino group. Synthesized by spirocyclization methodology, trans- and cis-diastereoisomers of target compounds were isolated and evaluated as potential anticancer and antimicrobial agents. Their molecular geometries were refined by ab initio minimizations. Pharmacophore modeling and QSAR studies were performed in order to correlate their molecular structure and biological activity.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/toxicidade , Indóis/síntese química , Indóis/toxicidade , Antineoplásicos/química , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Indóis/química , Modelos Químicos , Relação Quantitativa Estrutura-Atividade , Sesquiterpenos , Estereoisomerismo , Terpenos/síntese química , Terpenos/química , FitoalexinasRESUMO
Hypericin (Hyp) is a naturally occurring compound used as photosensitizer in photodynamic therapy and diagnosis. Recently, we have shown that Hyp presence alone, without illumination, resulted in substantial biological effects at several sub-cellular levels. Hyp induced changes in cellular ultrastructure, mitochondria function and metabolism, and distribution of Bcl2 proteins in malignant and non-malignant cells. The molecular mechanisms that underlie Hyp light-independent effects are still elusive. We have hypothesized that Bcl2-Hyp interactions might be one possible mechanism. We performed molecular docking studies to determine the Hyp-Bcl2 interaction profile. Based on the interaction profiles small Bcl2 peptide segments were selected for further study. We designed small peptides corresponding to Bcl2 BH3 and BH1 domains and tested the binding of Hyp and Bcl2 known inhibitor, ABT263, to the peptides in computer modeling and in vitro binding studies. We employed endogenous tryptophan and tyrosine in the BH3 and BH1 peptides, respectively, and their fluorescent properties to show interaction with Hyp and ABT263. Overall, our results indicate that Hyp can interact with Bcl2 protein at its BH3-BH1 hydrophobic groove, and this interaction may trigger changes in intracellular distribution of Bcl2 proteins. In addition, our computer modeling results suggest that Hyp also interacts with other anti-apoptotic members of Bcl2 family similar to the known BH3 mimetics. Our findings are novel and might contribute to understanding Hyp light-independent effects. In addition, they may substantiate the therapeutic use of Hyp as a BH3 mimetic molecule to enhance other cancer treatments.
Assuntos
Glioma/tratamento farmacológico , Perileno/análogos & derivados , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Compostos de Anilina/química , Compostos de Anilina/farmacologia , Antracenos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Fluorescência , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Perileno/química , Perileno/farmacologia , Fármacos Fotossensibilizantes/química , Sulfonamidas/química , Sulfonamidas/farmacologia , Triptofano/farmacologia , Tirosina/farmacologiaRESUMO
Atomic force microscopy, Thioflavin T (ThT) fluorescence assay, circular dichroism spectroscopy, differential scanning calorimetry, and molecular modeling techniques have been employed to investigate the amyloid aggregation of insulin in the presence of non-ionic detergent, Triton X-100 (TX-100). In contrast to recently described inhibition of lysozyme amyloid formation by non-ionic detergents (Siposova, 2017), the amyloid aggregation of insulin in the presence of sub-micellar TX-100 concentration exhibits two dissimilar phases. The first, inhibition phase, is observed at the protein to detergent molar ratio of 1:0.1 to 1:1. During this phase, the insulin amyloid fibril formation is inhibited by TX-100 up to â¼60%. The second, "morphological" phase, is observed at increasing detergent concentration, corresponding to protein:detergent molar ratio of â¼1:1 - 1:10. Under these conditions a significant increase of the steady-state ThT fluorescence intensities and a dramatically changed morphology of the insulin fibrils were observed. Increasing of the detergent concentration above the CMC led to complete inhibition of amyloidogenesis. Analysis of the experimental and molecular modeling results suggests an existence of up to six TX-100 binding sites within dimer of insulin with different binding energy. The physiological relevance of the results is discussed.
Assuntos
Amiloide/química , Proteínas Amiloidogênicas/química , Insulina/química , Octoxinol/química , Agregados Proteicos , Amiloide/metabolismo , Amiloide/ultraestrutura , Proteínas Amiloidogênicas/metabolismo , Benzotiazóis/química , Sítios de Ligação , Corantes Fluorescentes/química , Humanos , Concentração de Íons de Hidrogênio , Insulina/metabolismo , Cinética , Microscopia de Força Atômica , Simulação de Acoplamento Molecular , Octoxinol/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometria de Fluorescência , TermodinâmicaRESUMO
Natural polyphenols, curcumin, rottlerin and EGCG were selected for initial computational modeling of protein-ligand interaction patterns. The docking calculations demonstrated that these polyphenols can easily adjust their conformational shape to fit well into the binding sites of amyloidogenic proteins. The experimental part of the study focused on the effect of rottlerin on fibrillation of three distinct amyloidogenic proteins, namely insulin, lysozyme and Aß1-40 peptide. Different experimental protocols such as fluorescence spectroscopy, circular dichroism and atomic force microscopy, demonstrated that amyloid fibril formation of any of the three proteins is inhibited by low micromolar rottlerin concentrations. Most likely, the inhibition of amyloid formation proceeded via interaction of rottlerin with amyloidogenic regions of the studied proteins. Moreover, rottlerin was also effective in pre-formed fibrils disassembly, suggesting that interactions of rottlerin with fibrils were capable to interrupt the fibril-stabilizing bonds of ß-sheets. The apparent IC50 and DC50 values were calculated in the range of 1.3-36.4⯵M and 15.6-25.8⯵M, respectively. The strongest inhibiting/disassembling effect of rottlerin was observed on Aß1-40 peptide. The cytotoxicity assay performed on the Neuro 2a cells indicated time-dependent cell morphology changes but rottlerin affected the cell viability only at concentration above 50⯵M. The results of this study suggest that chemical modifications on rottlerin could be tested in the future as a promising strategy for the modulation of amyloidogenic proteins aggregation.
Assuntos
Acetofenonas/química , Peptídeos beta-Amiloides/química , Benzopiranos/química , Fragmentos de Peptídeos/química , Acetofenonas/farmacologia , Animais , Benzopiranos/farmacologia , Catequina/análogos & derivados , Catequina/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Curcumina/química , Insulina/química , Camundongos , Modelos Moleculares , Muramidase/químicaRESUMO
Insulin and lysozyme share the common features of being prone to aggregate and having biomedical importance. Encapsulating lysozyme and insulin in micellar nanoparticles probably would prevent aggregation and facilitate oral drug delivery. Despite the vivid structural knowledge of lysozyme and insulin, the environment-dependent oligomerization (dimer, trimer, and multimer) and associated structural dynamics remain elusive. The knowledge of the intra- and intermolecular interaction profiles has cardinal importance for the design of encapsulation protocols. We have employed various biophysical methods such as NMR spectroscopy, X-ray crystallography, Thioflavin T fluorescence, and atomic force microscopy in conjugation with molecular modeling to improve the understanding of interaction dynamics during homo-oligomerization of lysozyme (human and hen egg) and insulin (porcine, human, and glargine). The results obtained depict the atomistic intra- and intermolecular interaction details of the homo-oligomerization and confirm the propensity to form fibrils. Taken together, the data accumulated and knowledge gained will further facilitate nanoparticle design and production with insulin or lysozyme-related protein encapsulation.
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Two nonionic detergents, Triton X-100 (TX-100) and n-dodecyl-ß-d-maltoside (DDM) were tested for their ability to affect lysozyme amyloid aggregation. We have demonstrated that fibrillization of lysozyme is completely inhibited by low sub-micellar concentrations of both of these detergents. The apparent IC50 values were calculated to be 22µM and 26µM for TX-100 and DDM, respectively. The detergent/protein ratio is not the only parameter controlling inhibition. The precise timing of the detergent addition was found to be also crucial. It appears that the primary inhibitory activity of detergents resulted from inhibition of nuclei formation, in addition to inhibition of fibril polymerization at the early stage of protofibrils growth. The docking study revealed that Asn-59, Trp-63 and Ala-107, all present within the lysozyme amyloidogenic region, were involved in the interaction with both detergents. In addition, TX-100 also interacted with Gln-57 and Asp-103 within lysozyme. Moreover, based on our computational results, TX-100 bridges the Gln-57 and Ala-107 amino acids of the amyloidogenic segment of lysozyme and therefore inhibits more effectively the amyloid fibril formation. Along these lines, the knowledge gained from our study indicates that the detergents or their derivatives may be applicable as a promising strategy for the modulation of lysozyme protein aggregation.
Assuntos
Amiloide/química , Detergentes/química , Muramidase/química , Animais , Benzotiazóis , Sítios de Ligação , Galinhas , Dicroísmo Circular , Relação Dose-Resposta a Droga , Clara de Ovo/química , Glucosídeos/química , Humanos , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Cinética , Luz , Micelas , Microscopia de Força Atômica , Modelos Moleculares , Octoxinol/química , Conformação Proteica , Espalhamento de Radiação , Espectrofotometria Ultravioleta , Propriedades de Superfície , Temperatura , Tiazóis/químicaRESUMO
By means of fluorescence microscopy the intracellular distribution of fluorescent drugs with different hydrophobicity (quinizarin, emodin and hypericin) was studied. Selective photoactivation of these drugs in precisely defined position (nuclear envelope) allowed moderately hydrophobic emodin enter the nucleus. Highly hydrophobic hypericin was predominantly kept in the membranes with no fluorescence observed in the nucleus. The redistribution of quinizarin, emodin and hypericin between lipids, proteins and DNA was studied in solutions and cells. Based on these results was proposed theoretical model of hydrophobic drugs' nuclear internalization after photo-activation. Molecular docking models showed that hypericin has the strongest affinity to P-glycoprotein involved in the cell detoxification. Presence of 10 µM quinizarin, emodin or hypericin increased P-glycoprotein function in U87 MG cells. Moreover, emodin pretreatment allowed quinizarin nuclear internalization without photo-activation, which was not the case for hypericin. The synergy of such pretreatment and photo-activation should lessen the drug doses with simultaneous increase of drug efficacy triggering cell apoptosis/necrosis.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antraquinonas/farmacologia , Emodina/farmacologia , Perileno/análogos & derivados , Antracenos , Antraquinonas/química , Antraquinonas/efeitos da radiação , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Núcleo Celular/efeitos da radiação , LDL-Colesterol/química , DNA/química , Emodina/química , Emodina/efeitos da radiação , Glioma/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Luz , Simulação de Acoplamento Molecular , Perileno/química , Perileno/farmacologia , Perileno/efeitos da radiação , Albumina Sérica/químicaRESUMO
The Antimicrobial peptides (e.g. defensins, hevein-like molecules and food-protecting peptides like nisin) are able to interact specifically with contact structures on pathogen surfaces. Besides protein receptors, important recognition points for such contacts are provided by pathogen glycan chains or surface lipids. Therefore, structural data concerning surface exposed glycans and lipids are of the highest clinical interest since these recognition functions play a key role when optimising anti-infection therapies. Approaches in nanomedicine and nanopharmacology in which various biophysical techniques such as NMR (Nuclear Magnetic Resonance), AFM (Atomic Force Microscopy), SPR (Surface Plasmon Resonance) and X-ray crystallography can be combined with biochemical and cell-biological methods will lead to improved antimicrobial peptides by this rational drug design approach. Such a strategy is extremely well suited to support clinical studies focussing on an effective fight against multiresistant pathogens. The data sets which are described here can be considered as universal for the design of various antimicrobial drugs against certain pathogens (bacteria, viruses and fungi) which cause severe diseases in humans and animals. Furthermore, these insights are also helpful for progressing developments in the field of food conservation and food preservation. A detailed analysis of the structure-function relationships between antimicrobial peptides and contact molecules on pathogen surfaces at the sub-molecular level will lead to a higher degree of specificity of antimicrobial peptides.